gamma-Secretase is an unprecedented intramembrane-cleaving aspartyl protease necessary for life in multicellular organisms. It processes a broad and growing array of single-transmembrane proteins, including Notch, Delta/Jagged, CD44, cadherins and certain sodium channel subunits. gamma-Secretase was originally identified as the protease affecting the final scission of APP to release amyloid beta-protein, which is causally implicated in Alzheimer's disease (AD). Presenilin, mutations in which cause the most common and aggressive form of early-onset AD, contains two conserved intra-membrane aspartates that serve as the catalytic site. The gamma- secretase complex also contains 3 other integral membrane proteins: Nicastrin, Aph-1 and Pen-2. Because the protease complex contains 19 transmembrane domains, multiple glycosylation sites on Nicastrin, and are unstable in many relatively mild detergents, crystallographic approaches to its structure are difficult and remote. We recently purified the mammalian complex essentially to homogeneity and performed electron cryo- microscopy and single particle image analysis to obtain the first 3D map of the intramembrane protease. We now propose to build on this initial model by significantly improving the resolution of the structure. We will localize the catalytic site of the gamma-secretase complex, map the substrate initial docking site(s), and investigate if multiple docking sites exist on the gamma-secretase complex for different transmembrane substrates. This work should yield far more structural and mechanistic information on the unusual intramembrane enzyme, which is required for cell-fate decisions in all metazoans.